1. Field of the Invention
The invention relates to the chemical synthesis of oligonucleotides and to materials and processes that are useful in such synthesis.
2. Summary of the Related Art
Oligonucleotides have become indispensable tools in modern molecular biology, being used in a wide variety of techniques, ranging from diagnostic probing methods to PCR to antisense inhibition of gene expression. This widespread use of oligonucleotides has led to an increasing demand for rapid, inexpensive and efficient methods for synthesizing oligonucleotides. The synthesis of oligonucleotides for antisense and diagnostic applications can now be routinely accomplished. See e.g., Methods in Molecular Biology. Vol 20: Protocols for Oligonucleotides and Analogs pp. 165-189 (S. Agrawal, Ed., Humana Press, 1993); Oligonucleotides and Analogues: A Practical Approach pp. 87-108 (F. Eckstein, Ed., 1991); and Uhlmann and Peyman, supra. Agrawal and Iyer, Curr. Op. in Biotech. 6: 12 (1995); and Antisense Research and Applications (Crooke and Lebleu, Eds., CRC Press, Boca Raton, 1993). Early synthetic approaches included phosphodiester and phosphotriester chemistries. Khorana et al., J. Molec. Biol. 72: 209 (1972) discloses phosphodiester chemistry for oligonucleotide synthesis. Reese, Tetrahedron Lett. 34: 3143-3179 (1978), discloses phosphotriester chemistry for synthesis of oligonucleotides and polynucleotides. These early approaches have largely given way to the more efficient phosphoramidite and H-phosphonate approaches to synthesis. Beaucage and Caruthers, Tetrahedron Lett. 22: 1859-1862 (1981), discloses the use of deoxynucleoside phosphoramidites in polynucleotide synthesis. Agrawal and Zamecnik, U.S. Pat. No. 5,149,798 (1992), discloses optimized synthesis of oligonucleotides by the Hphosphonate approach.
Both of these modem approaches have been used to synthesize oligonucleotides having a variety of modified internucleotide linkages. Agrawal and Goodchild, Tetrahedron Lett. 28: 3539-3542 (1987), teaches synthesis of oligonucleotide methylphosphonates using phosphoramidite chemistry. Connolly et al., Biochemistry 23: 3443 (1984), discloses synthesis of oligonucleotide phosphorothioates using phosphoramidite chemistry. Jager et al., Biochemistry 27: 7237 (1988), discloses synthesis of oligonucleotide phosphoramidates using phosphoramidite chemistry. Agrawal et al., Proc. Natl. Acad. Sci. USA 85, 7079-7083 (1988), discloses synthesis of oligonucleotide phosphoramidates and phosphorothioates using H-phosphonate chemistry. Solid phase synthesis of oligonucleotides by any of the known approaches ordinarily involves the same generalized protocol. Briefly, this approach comprises anchoring the 3xe2x80x2-most nucleoside to a solid support functionalized with amino and/or hydroxyl moieties and subsequently adding the additional nucleosides in stepwise fashion. Desired internucleotide linkages are formed between the 3xe2x80x2 functional group (e.g., phosphoramidite group) of the incoming nucleoside and the 5xe2x80x2 hydroxyl group of the 5xe2x80x2-most nucleoside of the nascent, support-bound oligonucleotide.
Refinement of methodologies is still required, however, particularly when making a transition to large-scale synthesis (10 xcexcmol to 1 mmol and higher). See Padmapriya et al., Antisense Res. Dev. 4: 185 (1994). Several modifications of the standard phosphoramidite methods have already been reported to facilitate the synthesis and isolation of oligonucleotides. See e.g., Padmapriya et al., supra; Ravikumar et al., Tetrahedron 50: 9255 (1994); Theisen et al., Nucleosides and Nucleotides 12: 43 (1994); and Iyer et al., Nucleosides and Nucleotides 14: 1349 (1995) (Kuijpers et al., Nucl. Acids Res. 18: 5197 (1990); and Reddy et al., Tetrahedron Lett. 35: 4311 (1994).
One limitation in solid phase synthesis resides in the nature of the solid phase support upon which the oligonucleotide is synthesized. A variety of solid support materials have been described for solid phase oligonucleotide synthesis, the most prevalent of which is controlled-pore glass (CPG). (See, e.g., Pon, Methods in Molec. Biol. 20: 465 (1993)). Unfortunately, CPG suffers certain limitations that prevent it from being an ideal support material. See e.g., Ron et al., Biotechniques 6: 768 (1988); McCollum et al., Nucleosides and Nucleotides 6: 821 (1987); Bardella et al., Tetrahedron Lett. 31: 6231-6234 (1990) For example, CPG is unstable under the standard ammonium hydroxide procedure that is used to deprotect the oligonucleotide and to cleave it from the solid support.
To overcome these problems, various attempts have been made to develop polymer supports to replace CPG. See e.g., Gao et al., Tetrahedron Lett. 32: 5477-5479 (1991); The Gene Assembler(trademark), A Fully Automated DNA Synthesizer, Pharmacia Fine Chemicals, Uppsala, Sweden (1986). The use of organic supports in this context has been explored. Reddy et al., Tetrahedron Lett. 35: 5771-5774 (1994) discloses an organic support based on native Fractogel (xe2x80x9cToyopearlxe2x80x9d, TosoHaas, Philadelphia, Pa.). Fractogel, however, has inherent limitations as a support for oligonucleotide synthesis, due to its low density when packed in acetonitrile and its limited pore volume per unit bed volume. U.S. Pat. No. 5,668,268 discloses polymer supports that provide the efficiency that CPG provides without the deficiencies of CPG. However, the present inventors have discovered that certain solvents used for detritylation, notably methylene chloride, lead to channel current formation in these polymer supports.
There is, therefore, a need for processes for oligonucleotide synthesis that do not lead to channel current formation in these new supports. In addition, methylene chloride is a low boiling point chlorinated solvent, which makes its disposal problematic, and it is also expensive. Thus, there is also a need for processes for oligonucleotide synthesis that utilize cheaper solvents which are more readily disposed.
The invention provides processes for oligonucleotide synthesis that utilize cheaper solvents which are more readily disposed than the solvents of prior art processes, and which do not lead to channel current formation in organic polymeric supports.
In a first aspect, he invention provides an improved process for solid phase oligonucleotide synthesis. In this improved process according to the invention, the improvement comprises carrying out detritylation of the nascent oligonucleotide using an arene as solvent. In certain preferred embodiments of the process according to this aspect of the invention, such synthesis is carried out using the phosphoramidite, H-phosphonate, or phosphotriester approach. This process of oligonucleotide synthesis according to the invention detritylates nascent oligonucleotides at least as efficiently as processes utilizing methylene chloride as solvent, but is less expensive, creates less toxic wastes, and does not cause channel current formation in organic polymeric solid supports.
In a second aspect, the invention provides an improved process for oligonucleotide synthesis wherein the synthesis includes coupling and detritylation steps carried out on a passivated organic polymeric support. Passivation reduces the hydrophilic character of the particle surface and increases access for the hydrophobic reagents to the nascent oligonucleotide chain, thereby improving the efficiency of the synthesis.
The processes for synthesizing oligonucleotides according to the invention are useful for synthesizing oligonucleotides on a scale ranging from small laboratory scale to large commercial scale. Thus, the processes according to the invention can be used to supply oligonucleotides for research purposes, for diagnostic purposes and for therapeutic purposes.